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Setting Up for Köhler Illumination and Phase Contrast on the Olympus BH-2 Microscope Rev. 3 of 7

Setting Up for Köhler

Illumination and Phase Contrast on the Olympus BH-2 Microscope

Revision 3

Copyright © 2020 Carl Hunsinger

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Revision History

Revision Description of Changes Date

1 Initial release August 28, 2020

2 Added reference to green/yellow filters and bottom view of phase condenser. September 1, 2020

3 Clarified phase-annulus centering procedure. October 17, 2020


Table of Contents

Introduction and Scope ........................................................................................................................................................... 3

Setting Up for Köhler Illumination .......................................................................................................................................... 3

Set Phase Condenser to Brightfield Setting ........................................................................................................................ 3

Focus on Specimen Slide with Desired Objective ............................................................................................................... 3

Partially Close the Field Diaphragm .................................................................................................................................... 3

Focus the Condenser ........................................................................................................................................................... 4

Center the Condenser ......................................................................................................................................................... 4

Adjust Field Diaphragm to Fill the Field of View ................................................................................................................. 4

Setting Up for Phase-Contrast Illumination ............................................................................................................................ 5

Match Selector Dial with Low-Power Objective ................................................................................................................. 5

Replace Eyepiece with Phase-Centering Telescope ............................................................................................................ 5

Focus the Phase-Centering Telescope ................................................................................................................................ 5

Center the Phase Annulus ................................................................................................................................................... 6

Center the Remaining Phase Annuli ................................................................................................................................... 7

Replace Centering Telescope with Eyepiece ...................................................................................................................... 7

Place Green/Yellow Filter in Filter Receptacle .................................................................................................................... 7

How to Contact the Author ..................................................................................................................................................... 7

Using Phase Contrast .......................................................................................................................................................... 7

Periodically Verify Setup of Phase Annuli ........................................................................................................................... 7

Table of Figures

Figure 1 – Set selector dial for brightfield viewing ................................................................................................................. 3

Figure 2 – Specimen properly focused for viewing ................................................................................................................. 3

Figure 3 – Adjust the field diaphragm control ring ................................................................................................................. 3

Figure 4 – Field diaphragm partially closed ............................................................................................................................ 3

Figure 5 – Focus the spot using condenser focus knob .......................................................................................................... 4

Figure 6 – Illuminated spot properly focused ......................................................................................................................... 4

Figure 7 – Condenser centering thumbscrews ....................................................................................................................... 4

Figure 8 – Illuminated spot properly centered ....................................................................................................................... 4

Figure 9 – Properly centered and filling the view ................................................................................................................... 5

Figure 10 – Set condenser dial to match the objective .......................................................................................................... 5

Figure 11 – Replace right eyepiece with phase telescope ...................................................................................................... 5

Figure 12 – Phase telescope improperly focused ................................................................................................................... 5

Figure 13 – Adjust knurled focus ring to focus the image ...................................................................................................... 6

Figure 14 – Phase telescope properly focused ....................................................................................................................... 6

Figure 15 – Top view of BH2-PC/PCD condenser .................................................................................................................... 6

Figure 16 – Bottom view of BH2-PC/PCD condenser .............................................................................................................. 6

Figure 17 – Aperture ring on the BH2-CD condenser ............................................................................................................. 6

Figure 18 – Phase ring and annulus images ............................................................................................................................ 6

Figure 19 – Phase annulus properly centered ........................................................................................................................ 7

Figure 20 – Place green/yellow filter in filter receptacle ........................................................................................................ 7


Introduction and Scope This document describes the proper procedures to set up an Olympus BH-2 microscope for Köhler illumination and for phase-contrast viewing. The procedures described here were performed using an Olympus BHT microscope, but these procedures also apply to the BHS, BHSU, and BHTU models.

Setting Up for Köhler Illumination The following equipment will be needed to perform brightfield viewing on the Olympus BH-2 microscope:

• Olympus BH-2 microscope (BHS, BHSU, BHT, or BHTU) with condenser

• Olympus LB phase-contrast or brightfield objectives

Set Phase Condenser to Brightfield Setting If your scope is equipped with a Zernike-style phase-contrast condenser (BH2-PC or BH2-PCD), rotate the selector dial on the condenser until the number “0” is visible in the front of the dial. This is the correct dial position for brightfield viewing (see Figure 1).

Figure 1 – Set selector dial for brightfield viewing

Focus on Specimen Slide with Desired Objective Select the lowest-power objective on the microscope. Place a suitable specimen slide onto the stage and adjust the specimen positioning, lighting intensity, focusing, and condenser aperture setting (see Figure 16 and Figure 17) so that the specimen detail is clearly visible through the eyepieces. Following the usual sequence, work your way up to the objective that you wish to set up for Köhler illumination, repeating the above adjustments as necessary as you proceed through the objectives (see Figure 2).

Figure 2 – Specimen properly focused for viewing

Partially Close the Field Diaphragm With the specimen in sharp focus using the desired objective, partially close the field aperture diaphragm by turning the control ring counterclockwise until a reduced spot of illumination is visible through the eyepieces (see Figure 3 and Figure 4).

Figure 3 – Adjust the field diaphragm control ring

Figure 4 – Field diaphragm partially closed


Focus the Condenser Adjust the vertical position of the condenser, using the condenser focus knob (see Figure 5), until the outer perimeter of the illuminated spot seen in the visual field is sharply focused (see Figure 6). Note that most condenser types, except the BH2-AAC condenser, are poorly corrected for chromatic aberration, and if this is the case you will see color fringing on both sides of the critical focus point of the spot. If you experience this, just pick the intermediate position between equal amounts of red and blue fringing.

Figure 5 – Focus the spot using condenser focus knob

Figure 6 – Illuminated spot properly focused

Center the Condenser Next, rotate the field diaphragm control ring (see Figure 3), clockwise to increase the size of the illuminated spot, while also adjusting the centering of the condenser, using the two orthogonal centering thumbscrews on the condenser yoke (see Figure 7), repeating as necessary until the illuminated spot nearly

fills the entire field of view through the eyepieces and is well centered within the visual field (see Figure 8).

Figure 7 – Condenser centering thumbscrews

Figure 8 – Illuminated spot properly centered

Adjust Field Diaphragm to Fill the Field of View With the illuminated spot properly focused and centered within the field of view, rotate the field diaphragm control ring (see Figure 3) clockwise to increase the size of the illuminated spot such that the edge of the spot moves just beyond the borders of the visual field and can no longer be seen. If necessary, tweak the centering of the condenser with the two centering thumbscrews (see Figure 7) so that the illuminated spot remains centered as you make this final adjustment of the field diaphragm. You should now see the entire field of view with no evidence of the field diaphragm at all (see Figure 9). The microscope is


now properly configured for Köhler illumination using this objective. Note that the Köhler setup procedure must be repeated every time the objective is changed.

Figure 9 – Properly centered and filling the view

Setting Up for Phase-Contrast Illumination The following equipment will be needed to perform phase-contrast viewing on the Olympus BH-2 microscope: • Olympus BH-2 microscope (BHS, BHSU, BHT, or BHTU)

• BH2-PC or BH2-PCD Zernike-style phase-contrast condenser

• Olympus CT-5 or CT-30 phase-centering telescope (as appropriate)

• Olympus IF-550 (interference) or 45G533 (absorptive) filter

• One or more Olympus LB phase-contrast objectives

Match Selector Dial with Low-Power Objective Select the lowest-powered phase-contrast objective. Rotate the selector dial on the phase-contrast condenser until the number visible on the front of the dial matches the selected phase-contrast objective (see Figure 10).

Figure 10 – Set condenser dial to match the objective

Replace Eyepiece with Phase-Centering Telescope Carefully remove the right-most eyepiece and insert an Olympus phase-centering telescope1 in its place (see Figure 11).

Figure 11 – Replace right eyepiece with phase telescope

Focus the Phase-Centering Telescope Look into the phase-centering telescope. With the lighting intensity set to a comfortable viewing level, you should see a ring of light and a darker ring, similar to that shown in Figure 12. Note that the image may be so blurry that these features cannot be readily discerned. Also note that the relative positioning of the two rings may vary from that shown in Figure 12.

Figure 12 – Phase telescope improperly focused

1 “CT-5” for 23mm, and “CT-30” for 30mm tubes. Note that other manufacturer’s centering telescopes may be substituted for the Olympus CT, so long as the barrel diameter of the phase telescope is compatible with your ocular tubes.


Rotate the knurled focusing ring on the phase-centering telescope (see Figure 13) until the image seen through the telescope is sharply focused (see Figure 14).

Figure 13 – Adjust knurled focus ring to focus the image

Figure 14 – Phase telescope properly focused

Center the Phase Annulus Per the following procedure, carefully adjust the two orthogonal centering thumbscrews on the rear of the phase-contrast condenser (see Figure 15), in order to center the image of the phase annulus within the image of the phase ring in the objective (see Figure 18).

Figure 15 – Top view of BH2-PC/PCD condenser

Figure 16 – Bottom view of BH2-PC/PCD condenser

Figure 17 – Aperture ring on the BH2-CD condenser

Figure 18 – Phase ring and annulus images


To adjust the position of the phase annulus, carefully depress both centering thumbscrews such that the thumbscrews engage with the internal centering screws for the annulus, then adjust the thumbscrews as necessary to move the annulus into the proper position. Experiment with the effects of the two thumbscrews until you get a feel for how the centering mechanism works. Once you gain a feel for how this works, use both thumbscrews as necessary to adjust the position of the phase annulus so that its image falls within the image of the phase ring in the objective (see Figure 19). After the annulus has been properly centered, release the two centering thumbscrews and allow them to disengage from the internal centering screws for the phase annulus.

Never turn the thumbscrews counter-clockwise beyond the point where the annulus ceases to move, or clockwise beyond the point where the rotation becomes firm, and do not rotate the selector dial on the condenser while one or both centering thumbscrews are engaged with the internal centering screws for the phase annulus, otherwise damage to the phase-contrast condenser may result.

Figure 19 – Phase annulus properly centered

Center the Remaining Phase Annuli Repeat this same centering procedure to align the phase annuli in the condenser for the remaining phase-contrast objectives.

Replace Centering Telescope with Eyepiece Once the various phase annuli have been properly centered, remove and replace the phase-centering telescope with the original eyepiece.

Place Green/Yellow Filter in Filter Receptacle Carefully place the green/yellow phase-contrast filter (the IF-550 interference filter provides the best results, but lacking this, the 45G533 absorptive filter may be used instead) into the 45mm filter receptacle beneath the stage (see Figure 20). Congratulation, you have

now properly setup your BH-2 microscope for phase-contrast viewing.

Figure 20 – Place green/yellow filter in filter receptacle

Using Phase Contrast To use phase contrast, just be sure to always match the number on the selector dial of the phase-contrast condenser with the magnification of the selected phase-contrast objective. That is basically all there is to it. You will find that certain specimens are ideally suited for phase contrast, whereas others are more suited to regular brightfield viewing. Don’t forget to return the selector dial on the phase-contrast condenser to the “0” setting whenever you wish to switch back to regular brightfield viewing.

Note that to obtain optimal phase-contrast results, it is necessary to use the green/yellow filter in the illumination system, in order to provide light of the proper wavelengths to which the phase-contrast objectives were designed to operate. This filter is particularly important when perform photomicrography since that’s when image quality is most critical. The inevitable green/yellow cast that the filter imparts may be removed from the images by converting them to grayscale. However, for routine viewing, you may discover that you prefer to omit the filter from the illumination system, knowing that the phase-contrast effects will be less than ideal, but the color rendition will be much closer to normal.

Periodically Verify Setup of Phase Annuli It is good practice to re-check the centering of the phase annuli before starting a phase-contrast observation session.

How to Contact the Author Please feel free to direct any questions or comments regarding this document (or BH-2 microscopes in general) to the author at the following email address: [email protected]

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